Test for cancer employing esters of bile acid degradation products



United States Patent Ofihce 3,087,862 Patented Apr. 30, 1963 No Drawing.

This invention relates to a method of testing for cancer, and relates particularly to a serological cancer test employing a bile acid degradation product as the testing agent. This application is a continuation-in-part of application Serial Number 451,284, filed August 20, 1954, which is in turn a continuation-impart of Serial Number 368,009, (filed July 14, 1953, both now abandoned.

The effectiveness of cancer treatments depends to a great extent upon the early diagnosis of the disease. Accurate diagnosis of cancer in the very early stages, before any visible symptoms of the disease have appeared, is a very diflicult matter. A positive test for cancer in its early stages would, therefore, be of great value in the successful combating of the disease.

Accordingly, it is an object of this invention to provide an accurate test for cancer in its very early stages.

It is another object to provide a simple and rapid test for cancer.

A further object is to provide an accurate serological test for cancer.

The above and other objects will become apparent after reading the following specification.

Generally stated, the invention comprises treating blood serum from a person or other animal with an agent which will give an accurate indication as to whether or not the donor of the serum has cancer. It has been discovered that certain bile acid degradation products and esters of these products, when mixed under standardized conditions with a serum obtained from a person having a malignant new growth, will produce a characteristic flocculation reaction not observed when the serum is from a person not having a malignant growth. By degradation products of the bile acids and their esters are meant products obtained by such reactions as hydrolysis, reduction, dehydrogenation, oxidation, pyrolysis, dehydration, decarboxylation, and other reactions acting on the original bile acids or bile acid esters, but not destroying the ring structure of the cholane nucleus of the original acids or esters.

Furthermore, it has been found that the bile acid degradation product preferably is a degradation product of a bile acid such as cholic acid, desoxycholic acid, chenodesoxycholic acid and lithocholic acid, and particularly an ester of such degradation product. More speciiically stated, it has been found that the esters of cholenic acids, of choladienic acids and of cholatrienic acids may be used as serological cancer test agents as described above.

Of these esters, the lower esters, such as methyl and the ethyl esters, are readily made in high yields from the bile acids by dehydrating the esterified acid, and are desirable cancer test agents for this reason. Among the desirable test agents of the group are the esters of desoxycholenic acid, desoxycholadienic acid, and desoxycholatrienic acid.

Included among the cancer test agents for use in the method of this invention are partially dehydrated cholic acid, desoxycholic acid, chenodesoxycholic acid and lithocholic acid and their esters. That is, the removal of the hydroxyl groups in the molecule from the cholic acid, the desoxycholic acid and the chenodesoxycholic acid nuclei gives cholatrienic and choladienic acids and esters respectively. Apocholic acid and its esters, for

example, are included among these cancer test agents. One or both of the hydroxyl groups, as the case may be, of these hydroxy cholenic nuclei may be substituted by such radicals as halogen, sulfur, alkoxy, alkyl, substituted alkyl, amino, cyanogen, aldehyde, carboxyl, ester, amide, ketone, intro, and sul-fonic radicals for example. It has been found that the derivatives produced by substituting the hydroxyl radical in the 3-position of the hydroxy cholenic nucleus by a halogen atom, particularly the substitution of a chlorine atom for the hydroxyl in the 3-position of an ethyl ester of a 3-hydroxy cholenic acid, are relatively stabhe crystallin substances. 7

Compounds which have been found to be especially useful in the serological test of the invention are the lower alkyl esters of choladienic acid and 3-l3-chlorocholenic acid. A mixture of the ethyl esters of A and A -choladienic acid, such as that resulting from the dehydration and esterification of desoxycholic acid, has been found particularly suitable. The methyl, ethyl, capryl, isoamyl, and butyl esters of choladienic acid as well as the amide of such acid, give seroflocculation reactions. However, tests appear to indicate that the lower molecular weight esters are more satisfactory than those of relatively higher molecular weight. Methyl or ethyl esters of choladienic acid appear better suited to the test than the capryl or isoamyl esters, for example. Ethyl 3-[3-chloro-A -cholenate has also been found to be a satisfactory seroflocculation agent. A satisfactory method for producing these cancer test agents is described in my copending application Serial Number 451,284, filed August 20, 1954.

Compounds having the following general formula also are contemplated as serological test agents within the scope of the invention:

wherein R is an alkyl group and X, Y and Z are members of the group consisting of hydrogen, halogen, sulfur, alkoxy, alkyl, substituted alkyl, amino, cyanogen, aldehyde, carboxyl, ester, amide, ketone, nitro and sulfonic radicals.

Although the cancer test agent, when added to blood serum, will indicate the presence of cancer in the donor of the serum in various ways, it has been found convenient, when using certain of the cancer test agents, such as the esters of cholenic acids for example, to base the test on the character and presence or absence of flocculation or turbidity produced in the mixture of serium and cholenic acid ester. It is to be understood, however, that the invention comprehends the observance of other physical or chemical phenomena exhibited on addition of the test agent to the serum, and is not restricted to flocculation observance.

The preferred test procedure comprises separation of serum from the blood of a donor, inactivating such serum at a temperature of about 63 -65 C., mixing the inactivated serum with an antigen suspension containing the ester of the selected bile acid degradation product in a buffered saline solution, centrifuging the resulting mixture, and examining the resulting liquid for particle flocculation. A positive test result is indicated by the presence of particles in a clear liquid medium; a negative result is indicated by various degrees of turbidity. It has been found that 635 C. is the optimum temperature for inactivation of the blood serum, as inactivation of the serum at this temperature materially lessens the number of false positives. Inactivation at lower temperatures results in a greater number of false positives whereas higher temperatures tend to coagulate certain sera.

The following procedure illustrates a specific test method employing the ethyl ester of choladienic acid as the seroflocculation agent:

EXAMPLE 1 Blood is obtained by venipuncture under aseptic precautions and stored overnight at temperature between 4-6 C. Serum is separated from the clot by decantation and then centrifuged and heated for 30 minutes at 63.5 C. For testing, all sera and solutions must be maintained at 15 C. (iced water bath).

The following solutions are required: (A) Bulfered saline solution consisting of 6.5 ml. of 0.1 M citric acid, 43.5 ml. of 0.2 M anhydrous disodium phosphate, 42.5 ml. of 5% sodium chloride and then diluted to 250 ml. with water; (B) 1% cholesterol in absolute ethanol; (C) ethyl choladienate, 15 mg. per ml. in 95% ethanol.

The antigen suspension is prepared by depositing at the bottom of a tube 0.013 ml. of the cholesterol solu tion (B), plus 0.1 ml. of the antigen solution (ethyl choladienate solution (C) and mixing by rapid rotation of the tube. To the mixed alcoholic solutions B and C is added 0.4 ml. of the citrate phosphate buffered normal saline A. These are thoroughly mixed by gently inverting the tube several times. (Too rigorous agitation may precipitate suspension.) The suspension is used within 15 minutes after preparation.

The test is carried out as follows: All glassware is thoroughly cleansed, rinsed in distilled water and oven-dried. Two tubes (13 x 100 mm.) are used. To one is added .1 ml. of serum and .15 ml. of antigen suspension; to the other 0.07 ml. of serum and .15 ml. of antigen suspension. The tubes are shaken in a Kahn shaker (275 to 285 oscillations per minute, 1 /2" stroke), for 3 minutes. To each tube is then added .8 ml. of bufiered normal saline (A), and the tubes are then centrifuged at 3,000 r.p.m. for minutes. Readings are made in diffuse standardized light after shaking tubes to disperse precipitate at the bottom. Positive reactions give particles in a crystal clear medium in one or both tubes. Negative reactions give various degrees of turbidity with or without particles. If any doubt exists in interpretation, test is considered negative. A known positive and a known negative serum should be included in every test.

EXAMPLE 2 Serofiocculation tests utilizing the general procedure of Example 1 were performed on sera from a total number of 2747 individuals, consisting of 220 known to have cancer, 1822 normal, healthy individuals, chiefly bloodbank donors, and 705 individuals hospitalized with nonmalignant pathological conditions. The normal group of healthy individuals ranged in age from 20 to 70 years. Tests were run in duplicate, the sera being inactivated in one instance at 56 C. and in the other instance at 63.5 C. The antigen used was ethyl choladienate obtained from Lederle Laboratories. All sera studied were obtained from the Veterans Administration Hospital Center, Los Angeles, except the blood bank sera. The results of the tests are set forth in the following table:

It will be noted from Table I that the flocculation tests correctly indicated cancer in 204 out of the 220 known cases with the 56 C. inactivation, i.e., a total of 93%, and in 202 out of the 220 known cases with 63.5 C. inactivation, i.e., 92%. Tests on the normal group, including the donors of the blood bank sera as set forth in Table I, i.e., 1822 cases, demonstrate reduction of false positives from 13% to 0.6% by heating the serum to 635 C. However, it is clear that inactivation at the latter temperature did not diminish the sensitivity of the reaction with respect to the detection of malignancies. The occurrence of positive reactions in the nonmalignant hospitalized cases was also considerably diminished by conducting the tests with the 63.5 C. serum inactivation. The occurrence of positive reactions in this group is thought to be due to change in plasma similar to that which occurs in malignancies by reason of the particular pathological condition of the hospitalized patient.

Although the invention is herein shown and described in what is conceived to be the most practical and preferred embodiment, it is recognized that departures may be made therefrom' within the scope of the invention, which is not to be limited to the details disclosed herein but is to be accorded the full scope of the claim so as to embrace any and all equivalent means, test agents, and test methods.

I claim:

The method of testing the blood of a person for the presence of abnormalities in the person which comprises separating serum from the blood, inactivating said serum at a temperature within the range of about 63 to 65 C., mixing the inactivated serum with an antigen suspension containing ethyl choladienate in a buffered saline solution, centrifuging the resulting mixture, and determining the turbidity of the centrifuged mixture.

References Cited in the file of this patent Ma er: I. National Cancer Inst., June 1944, pp. 571- 578, especially page 572.

Cancer, vol. 33, January 1950, page 160.

Peacock and Williams: I. National Cancer Inst., vol. 18, No. 2, February 1957, pp. 277-283.

Hill: Proceedings Society of Experimental Biology and Medicine, March 1958, pp. 524-527. 

